Proteases represent an important class of regulatory enzymes, biotechnological reagents, and drug targets. Here we describe a fluorogenic biosensor for detecting protease activity, called Pro-Green Fluorescent Protein (Pro-GFP). Pro-GFP is a structural variant of split GFP that has been re-engineered to function specifically as a latent fluorophore. We report that Pro-GFP variants serve as robust fluorogenic substrates for five proteases, including thrombin, caspase-3 and HIV protease. These clinically-relevant, serine, cysteine and aspartic acid proteases, respectively, exhibit distinct substrate specificity and cleave by different mechanisms to activate Pro-GFP. In addition to protease monitoring in vitro, we show that Pro-GFP can be used as an in vivo reporter of protease activity in E. coli. The in vitro/in vivo versatility and the gain-of-fluorescence characteristic of Pro-GFP provide advantages not found in existing protease biosensors.
This preliminary program is subject to change.
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Cut and Glow: A Fluorogenic Biosensor for Proteases
Proteases represent an important class of regulatory enzymes, biotechnological reagents, and drug targets. Here we describe a fluorogenic biosensor for detecting protease activity, called Pro-Green Fluorescent Protein (Pro-GFP). Pro-GFP is a structural variant of split GFP that has been re-engineered to function specifically as a latent fluorophore. We report that Pro-GFP variants serve as robust fluorogenic substrates for five proteases, including thrombin, caspase-3 and HIV protease. These clinically-relevant, serine, cysteine and aspartic acid proteases, respectively, exhibit distinct substrate specificity and cleave by different mechanisms to activate Pro-GFP. In addition to protease monitoring in vitro, we show that Pro-GFP can be used as an in vivo reporter of protease activity in E. coli. The in vitro/in vivo versatility and the gain-of-fluorescence characteristic of Pro-GFP provide advantages not found in existing protease biosensors.